Upregulation of IL-6, IL-8 and CCL2 gene expression after acute inflammation: Correlation to clinical pain

Abstract

Tissue injury initiates a cascade of inflammatory mediators and hyperalgesic substances including prostaglandins, cytokines and chemokines. Using microarray and qRT-PCR gene expression analyses, the present study evaluated changes in gene expression of a cascade of cytokines following acute inflammation and the correlation between the changes in the gene expression level and pain intensity in the oral surgery model of tissue injury and acute pain. Tissue injury resulted in a significant upregulation in the gene expression of interleukin-6 (IL-6; 63.3-fold), IL-8 (8.1-fold), chemokine (C-C motif) ligand 2 (CCL2; 8.9-fold), chemokine (C-X-C motif) ligand 1 (CXCL1; 30.5-fold), chemokine (C-X-C motif) ligand 2 (CXCL2; 26-fold) and annexin A1 (ANXA1; 12-fold). The upregulation of IL-6 gene expression was significantly correlated to the upregulation of IL-8, CCL2, CXCL1 and CXCL2 gene expression. Interestingly, the tissue injury-induced upregulation of IL-6, IL-8 and CCL2 gene expression, was positively correlated to pain intensity at 3h post-surgery, the onset of acute inflammatory pain. However, ketorolac treatment did not have a significant effect on the gene expression of IL-6, IL-8, CCL2, CXCL2 and ANXA1 at the same time point of acute inflammation. These results demonstrate that the upregulation of IL-6, IL-8 and CCL2 gene expression contributes to the development of acute inflammation and inflammatory pain. The lack of effect of ketorolac on the expression of these gene products may be related to the ceiling analgesic effects of non-steroidal anti-inflammatory drugs.

Figure 1

Gene expression profile of inflammatory cytokines and receptors after tissue injury in a clinical model of acute inflammatory pain. The data derived from analysis using GE SuperArray are expressed as median (n = 9). *, p < 0.05 for comparison of pre-surgery post-surgery tissue (paired t-test). Insert: Representative images from SuperArrays (OHS-110) showing the up-regulation of inflammatory cytokines and receptor after acute inflammation. #, expression of housekeeping genes.

Figure 2

The changes in gene expression following acute inflammation and ketorolac treatment as assessed by qRT-PCR. The gene expression level is expressed as the average threshold cycle after normalization using 18 rRNA expression (Average Delt Ct). The open symbols represent the gene expression level from each subject in normal pre-surgery tissue and after tissue injury. The solid symbols represent mean ± SEM. *, p < 0.05; **, p<0.01, ***, p<0.001 significant difference from the pre-surgery group (paired t-test).

Figure 3

Correlation between gene expression level (RQ) and pain intensity (VAS). The association between the gene expression and pain scale was examined using Pearson correlation at the time of onset of clinical pain and was adjusted for age, sex and race.

Figure 4

Schematic diagram depicts the inflammatory cascade associated with tissue injury and acute inflammation and its relation to neutrophils and monocytes recruitment and to pain. The solid black lines indicate the data and information obtained from the current oral surgery model. The brown lines indicate the data and information obtained from previous studies; the solid lines indicate up-regulation and dashed lines indicate down-regulation. Tissue injury results in the up-regulation of prostagalndins, TNF-α, IL-1β, Il-6, ANXA1 and CYR61. IL-6 in turn up-regulates the chemokines IL-8, CCL2, CXCL1 and CXCL2, which results in the accumulation of leukocytes. IL-6, IL-8 and CCL2 contribute to the pain process at this early time point. Refer to the text for further details.